The dynamic localization of mitochondria is critical for neuronal viability and synaptic function, however the molecular machinery that directs mitochondrial transport remains largely unknown. Recent work from our lab identified Milton, a novel adaptor protein that associates with kinesin and mitochondria, as a necessary component for the transport of mitochondria into axons and its terminals. The focus of this proposal is to examine the functional relationship between Milton and Miro, an unconventional Rho-like GTPase that associates with mitochondria and binds to Milton. Miro is a transmembrane protein whose cytoplasmic region contains two GTPase domains and two calcium-binding EF-hands. The signaling domains and calcium-binding capacity of Miro could serve to direct mitochondrial transport via its regulation of mitochondrial association with the transport machinery. As a protein similarly necessary for mitochondrial [unreadable] localization in neurons, Miro is a candidate mitochondrial receptor for Milton. Through the use of cell biological, biochemical and in vivo genetic methods, Miro's function and interaction with Milton will be examined to decipher their role in the directed transport of mitochondria in neurons.